Testing for allergy ultimately constitutes an analysis of the activity of receptors of basophilic leucocytes and is thus referred to the area of application of the invention. In such testing, in most places, a practical working schedule with approximately the following appearance is used:
The patient has acquired a picture of his symptoms, which constitutes the base of the case history, which is noted by the physician. On the basis of his own experiences, the patient is often able to specify the allergen (e.g. grass or cat) and with special knowledge founded on knowledge of probable allergens in various environments, seasons etc the physician can obtain a fairly good picture of which allergens are of interest.
In order to objectify this there are existing tests.
These are of two types, one tests on the patient himself (in vivo tests), another tests on tissue samples, usually blood samples from the patient (in vitro tests). These are in brief as follows:
In vivo the reaction in question may be elicited by imitating the process which gives the symptom. Thus, the reaction may be provoked by the patient inhaling the allergen, whereby hay-fever and asthma attacks may be started. The allergen may be instilled into the eyes and cause running eyes etc.
The most common in vivo test is, however, skin prick tests, of which the most common is so-called prick test, where a drop of the allergen in solution or in powder form is applied to the skin whereupon a slender lancet is pricked through the allergen and underlying skin. After 20-30 minutes the result is read, which constitutes an itching blush of which the diameter is a rough measure of the degree of allergy. In a particular drop there is usually also added a reference substance allowing better judgement of the reaction (histamine).
The test is usually performed on the forearm, this to allow isolating of the reaction from the rest of the body if any vigourous reaction results. It is then easier to treat a possible generalization of the reaction (anaphylactic shock), which can be fatal.
In this way the so-called type 1 reaction (hay-fever, certain forms of asthma and rashes, red eyes and hypersensitivity towards certain drugs, etc) may be objectified.
In these tests, the allergen is allowed to diffuse into those cells which have receptors on the surface directed towards the allergen. These cells are the so-called mast cells in the tissues. Their counterpart is the blood vessel in the so-called basophilic leucocytes, which are present in a low number in comparison to other blood cells (around 0.5% of all leucocytes or white blood cells).
When the mast cell or the basophilic cell meets with an allergen under certain conditions, it empties its content of small granules, granula, from which then histamine is liberated and causes the symptoms as it reacts with histamine receptors on other cells.
A number of tests may also be executed in vitro in order to objectify or confirm an allergy.
The most common method in Sweden is PRIST.RTM.-test (Paper Radio Immune Sorbent Test, Pharmacia), wherein the concentration of IgE-molecules is measured. It is usually higher in allergic persons than in healthy persons. IgE are those molecules which constitute a part of the receptor, on the mast cells and basophilic leucocytes, for the allergen. IgE is formed by lymphocytes and particularly when these are stimulated by an allergen. Lymphocytes release allergens in the body fluids and a small portion of these get caught on the mast cells and basophilic leucocytes (also other cells in certain cases). Less than 0,1% of IgE is bound to the reacting basophilic leucocytes, but there is probably a very large variation in this among different individuals.
A positive PRIST does thus not mean that an allergic reaction is measured but that the fact is made use of that many, but not all, allergic persons have a higher concentration of IgE in the blood than healthy people. It is also possible to measure the concentration of specific IgE for a large number of allergens. The test is then called RAST.COPYRGT. (Pharmacia). Often there is a good correlation between RAST and a performed skin prick test, at least if the same allergen is used as the reagent of those tests.
These two tests are used to a varying extent to complete the case history and skin prick testing.
Relatively little blood is consumed for the Rast test, which utilizes radio logical technique, which is common technique of analysis in larger hospitals in Sweden. The cost for a test, however, is so high that most often only in vivo-test are carried out.
There are also other methods for in vitro tests for allergy. For research purposes the liberation of histamine or other substances from mast cells and basophilic leucocytes upon contact with allergens are used, but this methodology is complicated and expensive, with results which are often difficult to evaluate, since the proportion of histamine in the blood of healthy people can not yet be reliably determined and there are large difficulties in directly measuring in a blood sample, possible changes. In practically all described methods it has been necessary to first enrich and wash the cells before reaction, which then has to correlate with with the total histamine content of the cells. This requires large volumes of blood per allergen tested.
Especially in France, techniques have also been used where the basophilic leucocytes have been investigated in connection with allergy. In brief, the methodology has been approximately of the following type, which also applies if measurements on liberation of histamine have been made. A blood sample is divided into portions, possibly after a less complicated enrichment step. To each portion a specific proportion of allergen is added. Usually six portions containing some ml basophilic suspension are used to which a series of concentrations of allergen (to the extent as the reactive part of the allergen extract can be measured) from about 1 ng/ml in steps of a power of 10 to up to 1 mg/ml have been added. This varies to some extent with the allergen preparation, but a spectrum of 1 million concentration units was necessary to be included due to individual variations. After a waiting period of about half an hour the basophilic leucocytes were then counted in dried and stained blood smears in a microscope.
Due to the large amount of work, this has not had widespread use as a method for allergy tests, despite that the actual reaction of the effector cell in allergy disorder is measured on a sample of blood delivered to a hospital laboratory instead of being measured on the patient as in the skinprick-test.
Very recently, a couple of publications have described the use of high capacity cell counters in the evaluation of the reaction, but still the blood volume is large if many allergens are tested, since several portions are used for each allergen tested.